Heterotrimeric G Protein
Heterotrimeric guanine nucleotide-binding regulatory proteins (“heterotrimeric G proteins”) that transduce signals from cell surface receptors to intracellular effectors are composed of α, β and γ subunits. Gα subunits are grouped based on amino acid homology into four subfamilies: Gs, Gi, Gq and G12 (Strathman and Simon, Proc. Natl. Acad. Sci. (USA) 1991; 88:5582-5582). The α subunits possess intrinsic GTPase activity and belong to a much larger group of GTPases which share structural elements. In all of these GTPases, a cycle of guanine nucleotide exchange and hydrolysis enables the protein to exist in two distinct states. The cycle allows G proteins to transiently relay signals from cell-surface receptors to intracellular effectors. Upon interaction with the appropriate agonist, the receptor serves to accelerate the exchange of GDP for GTP on the G protein α subunit. The exchange is believed to be accompanied by dissociation of the α and β-γ subunits, allowing α(and in some cases β-γ) to interact with effectors. The intrinsic GTPase activity terminates the signal, returning the α subunit to its basal GDP-bound state. Studies have suggested that the G12 members α12 and α13 regulate signaling pathways involved in controlling cell growth and differentiation. See Vara Prasad et at, J. Biol. Chem. 1995, 270:18655-18659.
The full-length cDNAs encoding mouse Gα12 and Gα3, and the encoded translation products, are disclosed in Strathman and Simon, supra, the entire disclosure of which is incorporated herein by reference. The sequences have been deposited in the GenBank data base by the authors, accession nos. M63659 (Gα12) and M63660 (Gα13). Those GenBank records are incorporated herein by reference. The human Gα12 cDNA has also been cloned (Chan et at., Mol. Cell. Biol. 1993; 13:762-768, incorporated herein by reference).
When constitutively activated, GTP-binding proteins can induce neoplastic transformation. See Xu et al., Proc. Natl. Acad. Sci. (USA) 1993: 90:6741-6745, the entire disclosure of which is incorporated herein by reference. Overexpression of wild-type Gα12 in NIH 3T3 cells is weakly transforming; a GTPase-deficient mutant of Gα12 (Gα12Q229L) behaves as a potent oncogene and is highly transforming in NIH 3T3 cells (Xu et al., supra). It has been found that Gα2-transfected cells exhibit a remarkably increased level of arachidonic acid in response to serum, and this effect is observed in cells transfected with either wild-type or activated mutant Gα12 (Xu et al., supra).
The Gα12Q229L mutant results from insertion of a leucine residue at position 229 in lieu of glutamine in the wild-type Gα12 protein. The mutation occurs in a highly conserved region in G proteins which is involved in binding and GTPase activity. The Q229L mutation results in a GTPase-deficient form of Gα12. The mutation blocks GTPase activity so that the α subunit binds GTP and is constitutively active (Xu et al., supra; Dhanasekaran et al., J. Biol. Chem. 1993, 269:11802-11806; Jian et al., FEBS Left. 1993; 330:319-322; Vara Prasad et al., Oncogene 1994, 9:2425-2429; Voyno-Yasenetskaya et al., Oncogene 1994; 9:2559-2565).
COX-2
Cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) are the enzymes which convert arachidonic acid into prostaglandins. COX-1 is ubiquitously expressed and involved in cellular “housekeeping” functions of various tissues and organs. In contrast, COX-2 expression is rapidly induced in diverse cell types by different growth factors, mitogens, tumor promoters, and physiological stress stimuli. Transcriptional induction of COX-2 has been shown to be involved in different pathological conditions such as inflammation, pain, and fever. It has also been shown that the anti-inflammatory effects of aspirin and ibuprofen is through their inhibitory effect, albeit non-specific inhibitory effect, on COX-2. Furthermore, persistent activation of COX-2 has been shown to be associated with oncogenesis as well as the invasive potential of tumor cells.
Inhibitors of COX-2 are useful as therapeutics. See, for example, the discussion of the advantages of selective COX-2 inhibition set forth in U.S. Pat. No. 5,604,253.
What is needed is a simple, sensitive and rapid screening method for determining the COX-2 inhibitory activity of therapeutic candidates.